The search for genes active in HSC fate determination has not yet turned up candidate genes and candidate pathways that will explain HSC self-renewal. This project has 4 specific aims to address this issue. First, a highly reproducible method for assaying embryonic stem (ES) cell/lines in vitro and in vivo for hematopoietic and HSC fates has been developed and will be tested and optimized for multiple vs single gene transductions. Second, RT-PCR and immunostaining assays for expression of gene transcripts and gene products in each HSC subset (long-term [LT], short-term [ST], and nonself-renewing multipotent progenitors [MPP] at the single cell level will be used to screen known and yet-to-be identified candidate genes for biological testing. Third, the Li and Cohen dominant negative approach for gene discovery will be used in the ES cell assays described in the first aim to identify new candidates that regulate HSCs and hematopoiesis. Finally, an efficient, high- throughput gene transduced protocol derived from the first aim will be put in place to test the potential of candidate genes to modify HSC behavior, the candidate gene deriving from this project~s aims 2 and 3, and the other 3 projects in this PPG (Hood Clarke, Fuller).